WO2017080098A1 - Polyinosinic cell-amino compound-calcium chloride adjuvant and vaccine containing polyinosinic cell-amino compound-calcium chloride adjuvant - Google Patents

Polyinosinic cell-amino compound-calcium chloride adjuvant and vaccine containing polyinosinic cell-amino compound-calcium chloride adjuvant Download PDF

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WO2017080098A1
WO2017080098A1 PCT/CN2016/000544 CN2016000544W WO2017080098A1 WO 2017080098 A1 WO2017080098 A1 WO 2017080098A1 CN 2016000544 W CN2016000544 W CN 2016000544W WO 2017080098 A1 WO2017080098 A1 WO 2017080098A1
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adjuvant
amino
polyinosinic
amino compound
antibiotic
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林海祥
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林海祥
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/205—Rhabdoviridae, e.g. rabies virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants

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  • the present invention relates to the field of medical immunology and biology, and in particular to vaccine adjuvants and vaccines containing the same.
  • the composition of the novel adjuvant of the present invention a poly-muscle-amino compound-calcium chloride, abbreviated as a sulphate adjuvant and a vaccine containing a sulphate adjuvant.
  • the picocalcium phosphate adjuvant is a double-stranded RNA adjuvant
  • the current double-stranded RNA adjuvant includes PIC (polyriboinosinic polyribo-cytoidylic acid polyphosphonate polycytidine), PICLC (PIC with poly-L-lysine and carboxymethylcellulose polyinosine). Acid polycytidine-carboxymethylcellulose-lysine), PIC12U (PIC with uridylic acid in specific interval, PIC12 uracil trade name Ampiigen), PICKCa (PIC-kanmycin-Cacl2, polyinosinic polycytidine) Several acid-kanamycin-calcium chloride adjuvants.
  • the main problem solved by the present invention is that the difference between my original invention patent "polymyocyte complex immunoadjuvant and vaccine containing the adjuvant" is to remove the amino antibiotic substance such as kanamycin component in the original adjuvant and replace it with non-antibiotic. Amino compound, the other ingredients are unchanged.
  • Non-antibiotic amino compounds such as: spermine, spermidine, chitin (hexose), chitosan and other non-antibiotics contain polyamino compounds instead of kanamycin antibiotics, because they contain more amino groups, which are positive
  • the binding of the ions to the carboxyl anion in the poly-cell double-stranded ribonucleic acid stabilizes the double-stranded structure of the polymyocyte and removes the side effects of the above-mentioned amino antibiotic.
  • polyinosinic acid polycytidine polymeric myocyte
  • amino compound excluding an amino antibiotic
  • calcium ions calcium ions
  • the adjuvant is referred to as: a picocalcium adjuvant.
  • the Chinese compound is abbreviated as: poly-cell-amino compound-Cacl 2 .
  • a vaccine containing a calcium sulphate adjuvant according to 1.
  • Vaccines and tumor vaccines including intracellular infections are used for prevention and treatment. It is characterized in that the adjuvant is a polyinosinic acid polycytidine (polymeric myocyte) containing an amino compound (excluding an amino antibiotic) and a calcium ion.
  • the adjuvant is a polyinosinic acid polycytidine (polymeric myocyte) containing an amino compound (excluding an amino antibiotic) and a calcium ion.
  • a sulphate adjuvant as an immunopotentiator according to 1, non-specifically promoting the application of immunity. It is characterized in that the adjuvant is a polyinosinic acid polycytidine (polymeric myocyte) containing an amino compound (excluding an amino antibiotic) and a calcium ion.
  • PIC poly-cell
  • the amino compound contained in the arginine adjuvant is a polyamino substance which is positively charged and combines with the PIC polyanion to form a hydrogen bond.
  • the combination of forms stabilizes the double-strand structure of PIC, which can resist the degradation of nuclease to a certain extent, and is effective for human application.
  • non-antibiotic amino compounds such as: refined ammonia, spermidine, chitin (hexidine) Sugar), chitosan, etc. stabilize the double-strand structure of PIC and remove the side effects of amino antibiotics. Therefore, the sulphate adjuvant not only further increases the adjuvant effect but also is safer, and it also meets the relevant authorities' almost no vaccine or vaccine. Antibiotic regulations.
  • Amino compounds except aminoguanidine antibiotics, including, for example, oxyamino compounds, acyclic monoamines, polyamines and their derivatives and salts, sulfonic acid amino compounds, cycloalkanes, aromatic monoamines, aromatic polyamines And its derivatives and salts, amides.
  • oxyamino compounds including, for example, oxyamino compounds, acyclic monoamines, polyamines and their derivatives and salts, sulfonic acid amino compounds, cycloalkanes, aromatic monoamines, aromatic polyamines And its derivatives and salts, amides.
  • oxyamino compounds e.g., oxyamino compounds, acyclic monoamines, polyamines and their derivatives and salts, sulfonic acid amino compounds, cycloalkanes, aromatic monoamines, aromatic polyamines And its derivatives and salts, amides.
  • sulfonic acid amino compounds including, for example, oxya
  • chitin chitin
  • chitin (called after deacetylation) chitosan.
  • Chitin is a natural resource that is abundant in the earth and the only positively charged animal cellulose found in nature. Due to its unsaturated molecular group in the molecular structure, it has a strong adsorption effect on various negatively charged harmful substances. It can also eliminate the "garbage" in the human body and achieve the purpose of preventing disease and prolonging life. Because of its unique function, chitin is praised by European and American scientists as the sixth life element of the human body, which is as important as protein, fat, sugar, vitamins and minerals.
  • the molecular weight chitin is a high molecular weight substance with a molecular weight of up to 1 million. The higher the molecular weight, the stronger the adsorption capacity, which is suitable for industrial and environmental protection applications. Low molecular weight is easily absorbed by the body.
  • a chitosan having a molecular weight of about 7,000 contains about 30 glucosamine residues.
  • the deacetylated chitin is deacetylated to form chitosan.
  • Chitin is insoluble in water and cannot be used by the body because it is insoluble in acid and alkali. Deacetylation increases its solubility and is therefore absorbed by the body. When the N-acetyl group is removed by more than 55%, it is called chitosan.
  • the surface of the tumor brain has more negative charge than the surface of the normal cell, causing the surface charge imbalance of the cell, thus causing the adhesion between the cells to decrease and the tissue to be destroyed.
  • Positively charged polycationic electrolyte can adsorb to swollen The surface of the tumor cells neutralizes the charge, thereby inhibiting the growth and metastasis of the tumor cells.
  • hexa-glucosamine and “hexa-acetamidine” are all six bodies with a molecular weight of about 1,000 and are known to be key factors in anti-cancer.
  • Japanese therapeutic use of "chitosan” intravenous drug for the treatment of cancer is 5 times more effective than general anticancer drugs, that is, obtaining water-soluble derivative oligo from “chitosan", which is low in molecular weight of about 1,000.
  • Molecular weight substances the scientific community calls it "low molecular immunostimulating agent"!
  • chitin is the only positively charged animal cellulose on earth. Due to its unsaturated molecular group in the molecular structure, it has a strong adsorption effect on various negatively charged harmful substances.
  • the third generation of “chitosan” (chitin) is the latest low molecular immunostimulating agent. In terms of safety of use, in the case of rats, it is fatal to take 18 kilograms of "chitosan” per kilogram of body weight, and it can be seen that “chitosan” (chitin) is safer than edible sugar. Chitin does not directly inhibit cancer cells, but inhibits the action of cancer cells by activating the immune system. Therefore, in the new adjuvant of calcium sulphate, the substitution of chitin with chitin can be: 1.
  • Chitin has a plurality of positively charged amine groups, which are combined with polyanions of PIC which are macromolecules, and form more stable than kanamycin.
  • PIC double-stranded RNA structures are more effective in primates and humans. 2. High safety, and went to the side effects of antibiotics. 3. Can be taken orally, can be injected. 4. Similar to the adjuvant, it has enhanced immunity and anti-tumor effect.
  • chitosan also known as: chitosan chemical name: polyglucosamine (1-4)-2-amino-B-D
  • the molecular weight of the unit body is: 161.2
  • Chitosan is a product of chitin de-N-acetyl, and the chitosan sample can be calculated by a relatively simple intrinsic viscosity measurement.
  • Commercial chitosan has three different viscosities depending on its use, namely, high viscosity products of 0.7 to 1 Pa ⁇ s, medium viscosity products of 0.25 to 0.65 Pa ⁇ s, and low viscosity products of ⁇ 0.25 Pa ⁇ s.
  • the chitosan macromolecules have active hydroxyl groups and amino groups, and they have strong chemical reaction ability.
  • the chitosan molecule has a free amino group, which is easy to form a salt in an acidic solution and has a cationic nature.
  • Chitosan has a more pronounced amino group with the increase of the number of amino groups in its molecule, which is the unique property of it, which lays a foundation for many biological properties and processing characteristics of chitosan [1].
  • Chitosan can lower blood fat, lower blood sugar, prevent and control high blood pressure, can directly inhibit tumor cells, add 0.5mg/ml chitosan solution in a solution containing cancer cells, and all cancer cells die after 24 hours. It inhibits bacterial activity: Chitosan is easily dissolved in weak acid solvents. It is particularly worth noting that the dissolved solution contains amino groups (NH2+), which bind negative electrons to inhibit bacteria. Chitosan inhibits bacterial activity and makes it widely used in medicine, textiles and food. Chitosan and its derivatives have good antibacterial activity and can inhibit the growth and reproduction of some fungi, bacteria, and viruses.
  • chitosan As of 2013, there are three possible mechanisms: one is due to the polycation of chitosan, which is easy to act on the surface of the fungal cell with negatively charged groups, thus changing the fluidity and permeability of the pathogen cell membrane; Replication and transcription; the third is to block the metabolism of pathogens. Since 2010, many researchers have proposed that chitosan can achieve antibacterial goals by inducing pathogenesis-related proteins, accumulating secondary metabolites and signaling. The effect of water-soluble chitosan such as chitosan lactate, chitosan glutamate and chitosan hydrogenated glutamate on different bacterial cultures. It was found that chitosan lactate and chitosan glutamate have high antibacterial activity against Gram-positive bacteria and Gram-negative bacteria.
  • Chitosan has a series of biological effects that activate the body system and mediate the body system, and improve the system function of phagocytic cells.
  • the receptor for bacterial polysaccharides exists on the surface of macrophages, and chitosan, as an analogue of bacterial polysaccharides, stimulates the activation of macrophages, producing the following reactions: promoting its phagocytic function and enhancing its synergistic effect in other immune responses, Thereby, the body regulates T cells, NK cells and B cells, and mediates the cellular immune response and humoral immune response of the body. Therefore, chitosan has an immunomodulatory effect on the body.
  • Chitosan After taking chitosan, cancer patients can activate immune lymphocytes in the body to distinguish normal cells and cancer cells and kill cancer cells.
  • Chitosan can regulate the pH value in the body to weakly alkaline, improve the utilization of insulin, and help prevent and treat diabetes. In addition, it also has the function of regulating the endocrine system, which makes insulin secretion normal, inhibits blood sugar and lowers blood lipids.
  • chitosan or its derivative In the new preparation of sulphate calcium, the substitution of chitosan or its derivative for kanamycin can be: 1. From the above structural formula, it can be seen that chitosan or its derivative has multiple positively charged amino groups, and PIC is a macromolecule. The combination of polyanions forms a more stable PIC double-stranded RNA structure than kanamycin, and is more effective in primates and humans. 2. High safety, and went to the side effects of antibiotics. 3. Can be taken orally, can be injected. 4. Similar to the adjuvant, it has enhanced immunity and anti-tumor effect.
  • Polycyanoic acid (Poly C): available for purchase.
  • Poly inosine (Poly I): available for purchase.
  • Non-antibiotic amino compounds available for purchase
  • Calcium chloride can be purchased
  • Na 2 HPO 4 l2H 2 O 107.44 g was dissolved in 49 000 ml of 0.85% NaCl.
  • NaH 2 PO 4 .2H 2 O 0.936 g 107.44 g was dissolved in 1 000 ml of 0.85% NaCl.
  • Non-antibiotic amino compounds to be determined 1000
  • PBS was added to 40 000 ml, plus: 0.1 M Cacl 2 (Cangzhou Juhua Reagent Co., Ltd. 20060401): 2.222 g of 200 ml water for injection, and adjusted to pH 7.2 with about 100 ml of Na 2 HPO 4.12H 2 O. All solutes were dissolved, 45 ° C for 30 minutes, 0.1 M CaCl 2 was added dropwise, pH was adjusted, filtered through a 0.22 um filter, and dispensed.
  • Cacl 2 Roszhou Juhua Reagent Co., Ltd. 20060401
  • the content of the picocalcium adjuvant to be tested is intended to indicate the feasibility of the implementation, but it is not limited and cannot be changed.
  • test basis National concentrating cell injection test standard WS1-XG-050-2000
  • mice For further testing of toxicity, it is necessary to repeat 5-6 needles in mice for observation. Can't die, and should gain weight. Mouse allergy experiment.
  • the preparation and verification scheme of the rabies vaccine of squamous calcium adjuvant is exemplified, and the intention is to make people understand the feasibility of preparing the vaccine for the preparation of the squamous calcium adjuvant vaccine, which is not limited to the skin of calcium sulphate.
  • Adjuvant rabies vaccine includes all vaccines containing the adjuvant, particularly intracellular infection vaccines and tumor vaccines.
  • Test basis National Pharmacopoeia rabies purified vaccine manufacturing verification procedures and special requirements for serotonin adjuvant vaccine (to be determined)

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Abstract

Provided are a vaccine adjuvant and a vaccine containing the adjuvant, the adjuvant containing a polyinosinic cell-amino compound-calcium chloride, and the adjuvant itself also being a non-specific immunopotentiator.

Description

皮氨钙佐剂及含有皮氨钙佐剂的疫苗Sodium arginine adjuvant and vaccine containing sulphate adjuvant
本发明涉及医学免疫学和生物学领域，特别是涉及疫苗佐剂及含有该佐剂的疫苗。本发明的新佐剂的组成：聚肌胞-氨基化合物-氯化钙，简称皮氨钙佐剂以及含有皮氨钙佐剂的疫苗。The present invention relates to the field of medical immunology and biology, and in particular to vaccine adjuvants and vaccines containing the same. The composition of the novel adjuvant of the present invention: a poly-muscle-amino compound-calcium chloride, abbreviated as a sulphate adjuvant and a vaccine containing a sulphate adjuvant.
皮氨钙佐剂属双链RNA佐剂，目前双链RNA佐剂包括PIC(polyriboinosinic polyribo-cytoidylic acid聚肌苷酸聚胞苷酸)、PICLC(PIC with poly-L-lysine and carboxymethylcellulose聚肌苷酸聚胞苷酸-羧甲基纤维素-赖氨酸)、PIC12U(PIC with uridylic acid in specific interval，PIC12尿嘧啶商品名Ampiigen)、PICKCa(PIC-kanmycin-Cacl2，聚肌苷酸聚胞苷酸-卡那霉素-氯化钙佐剂)几种。由于灵长目以上动物血清核酸酶的水解作用，使PIC人体应用效果很差；PICLC人体应用有效但副作用大，PICl2U经过大量研究和临床试验还是在2012年被美国食品药品监督管理局(FDA)否决了。我原专利《聚肌胞复合物免疫佐剂及其疫苗》及《包含基于聚肌苷酸-聚胞苷酸的佐剂免疫物质》、《含基于聚肌苷酸-聚胞苷酸的佐剂粘膜免疫物质》专利都含有氨基抗生素类物质，尽管弥补了PIC、PICLC和PIC12U的不足，灵长类动物应用安全有效，但其中致命缺点——含有抗生素成分是有关国家药品监督管理局不允许的：1.规定在每剂人用疫苗中允许抗生素含量为50ng，而皮卡佐剂疫苗每剂可检测游离抗生素如卡那霉素达500ng以上。2.氨基抗生素副作用的耳毒性包括前庭功能损害和对听神经的损伤。3.由于氨基抗生素物质如卡那霉素分子含有2个氨基，与聚肌胞的氢键结合并不稳固，因此大部分是游离状态，影响在人体的佐剂效果并可能产生副作用。The picocalcium phosphate adjuvant is a double-stranded RNA adjuvant, and the current double-stranded RNA adjuvant includes PIC (polyriboinosinic polyribo-cytoidylic acid polyphosphonate polycytidine), PICLC (PIC with poly-L-lysine and carboxymethylcellulose polyinosine). Acid polycytidine-carboxymethylcellulose-lysine), PIC12U (PIC with uridylic acid in specific interval, PIC12 uracil trade name Ampiigen), PICKCa (PIC-kanmycin-Cacl2, polyinosinic polycytidine) Several acid-kanamycin-calcium chloride adjuvants. Due to the hydrolysis of animal nucleases in primates, PIC human body application is very poor; PICLC human body is effective but has a large side effect. PICl2U has been rejected by the US Food and Drug Administration (FDA) in 2012 after extensive research and clinical trials. . My original patent "Polymerocyte complex immunoadjuvant and its vaccine" and "adjuvant immunoassay based on polyinosinic acid-polycytidine", "containing polyinosinic acid-polycytidine-based adjuvant The mucosal immune substances patent contains amino antibiotics. Although it makes up for the shortage of PIC, PICLC and PIC12U, primate applications are safe and effective, but the fatal shortcomings - containing antibiotics are not allowed by the relevant drug regulatory authorities. 1. The requirement is that the antibiotic content is 50 ng per dose of human vaccine, and the Picca adjuvant vaccine can detect free antibiotics such as kanamycin up to 500 ng per dose. 2. Ototoxicity of side effects of amino antibiotics includes vestibular dysfunction and damage to the auditory nerve. 3. Since amino antibiotic substances such as kanamycin molecules contain two amino groups, the hydrogen bond with the polymyocytes is not stable, and therefore most of them are in a free state, affecting the adjuvant effect in the human body and may cause side effects.
本发明主要解决的问题和我的原发明专利《聚肌胞复合物免疫佐剂及含有该佐剂的疫苗》的不同是去掉原佐剂中氨基抗生素物质如卡那霉素成分换成非抗生素类氨基化合物，其它成分不变。非抗生素氨基化合物，如：精胺、亚精胺、甲壳素(己丁糖)、壳聚糖等非抗生素含有多氨基化合物代替卡那霉素类抗生素物质，因含有更多氨基，其中的正离子与聚肌胞双链核糖核酸中的羧基负离子结合更能稳定聚肌胞的双链结构并去掉上述氨基类抗生素的副作用。The main problem solved by the present invention is that the difference between my original invention patent "polymyocyte complex immunoadjuvant and vaccine containing the adjuvant" is to remove the amino antibiotic substance such as kanamycin component in the original adjuvant and replace it with non-antibiotic. Amino compound, the other ingredients are unchanged. Non-antibiotic amino compounds, such as: spermine, spermidine, chitin (hexose), chitosan and other non-antibiotics contain polyamino compounds instead of kanamycin antibiotics, because they contain more amino groups, which are positive The binding of the ions to the carboxyl anion in the poly-cell double-stranded ribonucleic acid stabilizes the double-stranded structure of the polymyocyte and removes the side effects of the above-mentioned amino antibiotic.
为此本发明提供的技术方案是：To this end, the technical solution provided by the present invention is:
1.一种含氨基化合物(除外氨基类抗生素)和钙离子的聚肌苷酸聚胞苷酸(聚肌胞)在制备免疫佐剂中的应用。该佐剂简称：皮氨钙佐剂。成分中文简写为：聚肌胞-氨基化合物-Cacl2。1. Use of polyinosinic acid polycytidine (polymeric myocyte) containing an amino compound (excluding an amino antibiotic) and calcium ions in the preparation of an immunoadjuvant. The adjuvant is referred to as: a picocalcium adjuvant. The Chinese compound is abbreviated as: poly-cell-amino compound-Cacl 2 .
2.根据1所述的一种含有皮氨钙佐剂的疫苗。包括细胞内感染(病毒和细胞内感染的细菌、寄生虫等)疫苗和肿瘤疫苗用于预防和治疗。其特征在于所述的佐剂在于含有氨基化合物(除外氨基类抗生素)和钙离子的聚肌苷酸聚胞苷酸(聚肌胞)。2. A vaccine containing a calcium sulphate adjuvant according to 1. Vaccines and tumor vaccines including intracellular infections (bacteria, parasites, etc. of viruses and intracellular infections) are used for prevention and treatment. It is characterized in that the adjuvant is a polyinosinic acid polycytidine (polymeric myocyte) containing an amino compound (excluding an amino antibiotic) and a calcium ion.
3.一种含氨基化合物(除外氨基类抗生素)和钙离子的聚肌苷酸聚胞苷酸(聚肌胞)的免疫佐剂在制备用于哺乳动物或人的预防性和治疗性疫苗中的应用。3. An immunoadjuvant of polyinosinic acid polycytidine (polymeric myocyte) containing an amino compound (excluding an amino antibiotic) and calcium ions in the preparation of a prophylactic and therapeutic vaccine for mammals or humans Applications.
4.根据1所述用含有皮氨钙佐剂的免疫刺激物体外刺激免疫细胞，回输体内，已达到增强免疫抗肿瘤、抗细胞内感染效果的应用。其特征在于所述的佐剂在于含有氨基化合物(除外氨基类抗生素)和钙离子的聚肌苷酸聚胞苷酸(聚肌胞)。 4. The immune cell stimulated by an immunostimulating substance containing arginine adjuvant according to 1, and is returned to the body, and has been applied to enhance the anti-tumor and anti-intracellular infection effects of the immune system. It is characterized in that the adjuvant is a polyinosinic acid polycytidine (polymeric myocyte) containing an amino compound (excluding an amino antibiotic) and a calcium ion.
5.根据1所述用皮氨钙佐剂为免疫增强剂，非特异促进机体免疫力的应用。其特征在于所述的佐剂在于含有氨基化合物(除外氨基类抗生素)和钙离子的聚肌苷酸聚胞苷酸(聚肌胞)。5. The use of a sulphate adjuvant as an immunopotentiator according to 1, non-specifically promoting the application of immunity. It is characterized in that the adjuvant is a polyinosinic acid polycytidine (polymeric myocyte) containing an amino compound (excluding an amino antibiotic) and a calcium ion.
本发明的机制和效果是：PIC(聚肌胞}是大分子的聚阴离子，皮氨钙佐剂中含有的氨基化合物是多氨基物质，带正电荷，与PIC聚阴离子结合，形成了氢键形式的结合，稳定了PIC的双链结构，可以在一定程度上抵抗核酸酶的降解，而使人体应用有效。我们选择非抗生素氨基化合物，如：精氨、亚精氨、甲壳素(己丁糖)、壳聚糖等稳定PIC的双链结构并去掉氨基抗生素的副作用。因此皮氨钙佐剂不仅进一步增加了佐剂效果并且更加安全，也符合有关当局在疫苗、菌苗中几乎不能含有抗生素的规定。The mechanism and effect of the present invention are: PIC (poly-cell) is a polyanion of a macromolecule, and the amino compound contained in the arginine adjuvant is a polyamino substance which is positively charged and combines with the PIC polyanion to form a hydrogen bond. The combination of forms stabilizes the double-strand structure of PIC, which can resist the degradation of nuclease to a certain extent, and is effective for human application. We choose non-antibiotic amino compounds, such as: refined ammonia, spermidine, chitin (hexidine) Sugar), chitosan, etc. stabilize the double-strand structure of PIC and remove the side effects of amino antibiotics. Therefore, the sulphate adjuvant not only further increases the adjuvant effect but also is safer, and it also meets the relevant authorities' almost no vaccine or vaccine. Antibiotic regulations.
氨基化合物(氨基甙类抗生素除外)，包括如：含氧基氨基化合物，无环单氨、多氨及其衍生物和盐，磺酸类氨基化合物，环烷氨、芳香单氨、芳香多氨及其衍生物和盐，酰氨类化合物。具体如精氨、亚精氨、甲壳素(己丁糖)、壳聚糖、氨基葡萄糖等。Amino compounds (except aminoguanidine antibiotics), including, for example, oxyamino compounds, acyclic monoamines, polyamines and their derivatives and salts, sulfonic acid amino compounds, cycloalkanes, aromatic monoamines, aromatic polyamines And its derivatives and salts, amides. Specifically, such as refined ammonia, spermidine, chitin (hexose), chitosan, glucosamine and the like.
下面以甲壳素(己丁糖)、壳聚糖为例，阐述非抗生素类氨基化合物性质、作用和作为新佐剂成分应用的可行性，目的是结合实例说明本发明作用机理、可行性及其人用的安全性，并非仅限定这两种非抗生素氨基化合物，而在帮助理解，对本发明的保护范围作出更清楚的界定。The following is an example of chitin (hexose) and chitosan to illustrate the properties and functions of non-antibiotic amino compounds and the feasibility of application as a new adjuvant component. The purpose is to illustrate the mechanism, feasibility and feasibility of the invention with examples. The safety of human use is not limited to only these two non-antibiotic amino compounds, but to help understand, the scope of protection of the present invention is more clearly defined.
一、非抗生素氨基化合物举例：1. Examples of non-antibiotic amino compounds:
1.甲壳素Chitin
一般通称：甲壳质，甲壳素，(经脱乙酰化后称为)壳聚糖.Generally known as: chitin, chitin, (called after deacetylation) chitosan.
英文名称：Chitin.English name: Chitin.
中文学名：几丁质、甲壳素[2]Chinese name: chitin, chitin [2]
化学名称：β-(1，4)-2-乙酰氨基-2-脱氧-D-葡萄糖Chemical name: β-(1,4)-2-acetamido-2-deoxy-D-glucose
别名：壳多糖、几丁质、甲壳质、明角质、聚乙酰氨基葡糖Alias: chitin, chitin, chitin, keratin, acetyl glucosamine
分子式：(C8H13NO5)nMolecular formula: (C8H13NO5)n
性状：外观为类白色无定形物质，无臭、无味。Character: Appearance is white-like amorphous substance, odorless and tasteless.
甲壳素是地球上存量极为丰富的一种自然资源，也是自然界中迄今为止被发现的唯一带正电荷的动物纤维素。由于它的分子结构中带有不饱和的阳离子基团，因而对带负电荷的各类有害物质具有强大的吸附作用。同样它也能清除人体内的“垃圾”，达到预防疾病、延年益寿的目的。由于甲壳素具有这种独特功能，它被欧美科学家誉为和蛋白质、脂肪、糖类、维生素、矿物质同等重要的人体第六生命要素。Chitin is a natural resource that is abundant in the earth and the only positively charged animal cellulose found in nature. Due to its unsaturated molecular group in the molecular structure, it has a strong adsorption effect on various negatively charged harmful substances. It can also eliminate the "garbage" in the human body and achieve the purpose of preventing disease and prolonging life. Because of its unique function, chitin is praised by European and American scientists as the sixth life element of the human body, which is as important as protein, fat, sugar, vitamins and minerals.
分子量甲壳质是高分子量物质，其分子量可达100万以上。分子量越高吸附能力越强，适合工业、环保领域应用。低分子量容易被人体吸收。分子量为7000左右的几丁聚糖，大约含30个左右的葡萄糖胺残基。The molecular weight chitin is a high molecular weight substance with a molecular weight of up to 1 million. The higher the molecular weight, the stronger the adsorption capacity, which is suitable for industrial and environmental protection applications. Low molecular weight is easily absorbed by the body. A chitosan having a molecular weight of about 7,000 contains about 30 glucosamine residues.
脱乙酰基纯度几丁质经过脱乙酰基成为几丁聚糖。几丁质因为不溶于酸碱也不溶于水而不能被身体利用。脱乙酰基后可增加其溶解性因此可被身体吸收。N-乙酰基脱去55％以上的则称为壳聚糖。The deacetylated chitin is deacetylated to form chitosan. Chitin is insoluble in water and cannot be used by the body because it is insoluble in acid and alkali. Deacetylation increases its solubility and is therefore absorbed by the body. When the N-acetyl group is removed by more than 55%, it is called chitosan.
甲壳素功用：Chitin function:
研究发现，肿瘤细脑表面比正常细胞表面具有更多的阴电荷.造成细胞表面电荷不平衡，于是使细胞之间粘附力下降，组织迈破坏。带阳电荷的聚阳离子电解质能吸附到肿 瘤细胞的表面并使电荷中和，从而抑制了肿瘤细胞的生长和转移。The study found that the surface of the tumor brain has more negative charge than the surface of the normal cell, causing the surface charge imbalance of the cell, thus causing the adhesion between the cells to decrease and the tissue to be destroyed. Positively charged polycationic electrolyte can adsorb to swollen The surface of the tumor cells neutralizes the charge, thereby inhibiting the growth and metastasis of the tumor cells.
附图1.甲壳素抗癌医学图Figure 1. Chitin anticancer medical chart
这两种特定性代谢物：″己-葡萄氨″和″己-乙醯葡萄氨″均是分子量约1,000的六体，确知是抗癌瘤的关键因素。日本治疗癌应用″几丁聚糖″静脉注射药物，效果超过一般抗癌药物达5倍之多，即获取来自″几丁聚糖″的水溶性衍生物寡，它们是属于分子量约1,000的低分子量物质，科学界称之为″低分子免疫赋活剂″！These two specific metabolites: "hexa-glucosamine" and "hexa-acetamidine" are all six bodies with a molecular weight of about 1,000 and are known to be key factors in anti-cancer. Japanese therapeutic use of "chitosan" intravenous drug for the treatment of cancer is 5 times more effective than general anticancer drugs, that is, obtaining water-soluble derivative oligo from "chitosan", which is low in molecular weight of about 1,000. Molecular weight substances, the scientific community calls it "low molecular immunostimulating agent"!
因此，在上述癌细胞溶液中加入同样浓度的甲壳素，24小时后癌细胞的存活率达94％。这是体外试验的情况，在体内试验，就可见到甲壳素发挥的作用。试验是在鼠的腹腔内移植个艾氏腹水癌细胞或肿瘤细胞，在移植癌细胞或肿瘤细胞前的第6、第4和第2的三天，投喂精制甲壳素或卡介苗.共观察60天。试验结果表明，每天按每千克体重投喂50mg甲壳质，病鼠的治愈率都是67％，对照组却全都死亡，体内试验，甲壳素即能显示出对癌细胞或肿瘤细胞的治疗效果.在体外试验中却没有，这就说明了甲壳素不带阳电荷。对癌细胞或肿瘤细胞不起聚集粘附作用和电荷中和作用，所以不能直接抑制这些细胞，但能通过活性免疫***达到抑制癌细胞的目的.Therefore, the same concentration of chitin was added to the above cancer cell solution, and the survival rate of the cancer cells was 94% after 24 hours. This is the case of an in vitro test, and in vivo, the effect of chitin is seen. The test was to transplant an Ehrlich ascites cancer cell or tumor cell in the abdominal cavity of the mouse, and to deliver purified chitin or BCG in the third, fourth and second days before transplantation of the cancer cell or tumor cell. day. The test results showed that 50 mg of chitin was administered per kilogram of body weight per day, and the cure rate of the diseased mice was 67%. The control group all died. In vivo, chitin could show the therapeutic effect on cancer cells or tumor cells. Not in the in vitro test, this indicates that chitin is not positively charged. It does not inhibit aggregation and charge neutralization of cancer cells or tumor cells, so it cannot directly inhibit these cells, but it can achieve the purpose of inhibiting cancer cells through the active immune system.
可见，通过对甲壳素的不断认识和研究，它会发挥重大作用，对人类的生命延续起到积极的作用。It can be seen that through the continuous understanding and research of chitin, it will play a major role and play a positive role in the continuation of human life.
甲壳素作为新佐剂成分的可行性：Feasibility of chitin as a new adjuvant ingredient:
由于甲壳素是地球上唯一带正电荷的动物纤维素。由于它的分子结构中带有不饱和的阳离子基团，因而对带负电荷的各类有害物质具有强大的吸附作用。第三代″几丁聚糖″(甲壳素)就属于最新的低分子免疫赋活剂。在使用安全性方面，以老鼠为例，每公斤体重需服入18公斤″几丁聚糖″方可致死，可见″几丁聚糖″(甲壳素)比食用砂糖更安全。甲壳素不能直接抑制癌细胞，而是通过活化免疫***显示抑制癌细胞的作用。因此，在皮氨钙新佐剂中以甲壳素取代卡那霉素可以：1.甲壳素带多个正电荷胺基，与PIC是大分子的聚阴离子结合，形成较卡那霉素更稳定PIC双链RNA结构，在灵长类和人体中更有效。2.安全性高，又去到了抗生素副作用。3.可口服，可注射。4.与佐剂性质相似，具有增强免疫，抗肿瘤功效。Because chitin is the only positively charged animal cellulose on earth. Due to its unsaturated molecular group in the molecular structure, it has a strong adsorption effect on various negatively charged harmful substances. The third generation of "chitosan" (chitin) is the latest low molecular immunostimulating agent. In terms of safety of use, in the case of rats, it is fatal to take 18 kilograms of "chitosan" per kilogram of body weight, and it can be seen that "chitosan" (chitin) is safer than edible sugar. Chitin does not directly inhibit cancer cells, but inhibits the action of cancer cells by activating the immune system. Therefore, in the new adjuvant of calcium sulphate, the substitution of chitin with chitin can be: 1. Chitin has a plurality of positively charged amine groups, which are combined with polyanions of PIC which are macromolecules, and form more stable than kanamycin. PIC double-stranded RNA structures are more effective in primates and humans. 2. High safety, and went to the side effects of antibiotics. 3. Can be taken orally, can be injected. 4. Similar to the adjuvant, it has enhanced immunity and anti-tumor effect.
2.壳聚糖2. Chitosan
外文名：chitosan又称：脱乙酰甲壳素化学名：聚葡萄糖胺(1-4)-2-氨基-B-DForeign name: chitosan also known as: chitosan chemical name: polyglucosamine (1-4)-2-amino-B-D
化学名：β-(1→4)-2-氨基-2-脱氧-D-葡萄糖Chemical name: β-(1→4)-2-amino-2-deoxy-D-glucose
分子式：(C6H11NO4)NMolecular formula: (C6H11NO4)N
单元体的分子量为：161.2The molecular weight of the unit body is: 161.2
结构式如下式Structural formula
Figure PCTCN2016000544-appb-000001
Figure PCTCN2016000544-appb-000001
应用：医药、食品、化工、化妆品Application: medicine, food, chemical, cosmetics
壳聚糖是甲壳素脱N-乙酰基的产物，壳聚糖样品通过比较简单的特性粘度测量，即可计算其平均分子量。商品壳聚糖视其用途不同有三种不同的粘度，即高粘度产品为0.7～1Pa·s、中粘度产品为0.25～0.65Pa·s、低粘度产品＜0.25Pa·s。Chitosan is a product of chitin de-N-acetyl, and the chitosan sample can be calculated by a relatively simple intrinsic viscosity measurement. Commercial chitosan has three different viscosities depending on its use, namely, high viscosity products of 0.7 to 1 Pa·s, medium viscosity products of 0.25 to 0.65 Pa·s, and low viscosity products of <0.25 Pa·s.
壳聚糖大分子中有活泼的羟基和氨基，它们具有较强的化学反应能力。壳聚糖分子中带有游离氨基，在酸性溶液中易成盐，呈阳离子性质。壳聚糖随其分子中含氨基数量的增多，其氨基特性越显著，这正是其独特性质的所在，由此奠定了壳聚糖的许多生物学特性及加工特性的基础[1]。The chitosan macromolecules have active hydroxyl groups and amino groups, and they have strong chemical reaction ability. The chitosan molecule has a free amino group, which is easy to form a salt in an acidic solution and has a cationic nature. Chitosan has a more pronounced amino group with the increase of the number of amino groups in its molecule, which is the unique property of it, which lays a foundation for many biological properties and processing characteristics of chitosan [1].
壳聚糖能降血脂、降血糖、预防和控制高血压，能直接抑制肿瘤细胞，在含有癌细胞的溶液中.加入0.5mg/ml壳聚糖溶液，24小时后癌细胞全部死亡。能抑制细菌活性：壳聚糖在弱酸溶剂中易于溶解，特别值得指出的是溶解后的溶液中含有氨基(NH2+)，这些氨基通过结合负电子来抑制细菌。壳聚糖的抑制细菌活性，使其在医药、纺织和食品等领域有着广泛的应用。壳聚糖及其衍生物有较好的抗菌活性，能抑制一些真菌、细菌、和病毒的生长繁殖。截止到2013年认为其可能的机制有三：一是由于壳聚糖的多聚阳离子，易于真菌细胞表面带负电荷的基团作用，从而改变病原菌细胞膜的流动性和通透性；二是干扰DNA的复制与转录；三是阻断病原菌代谢。2010年以来，有许多研究者提出壳聚糖通过诱导病程相关蛋白，积累次生代谢产物和信号传导等方式来达到抗菌的目的的观点。水溶性壳聚糖如壳聚糖乳酸盐、壳聚糖谷氨酸盐和壳聚糖氢化谷氨酸盐对不同细菌培殖的影响。结果发现，壳聚糖乳酸盐和壳聚糖谷氨酸盐对革兰氏阳性菌和革兰氏阴性菌都有较高的抗菌作用。Chitosan can lower blood fat, lower blood sugar, prevent and control high blood pressure, can directly inhibit tumor cells, add 0.5mg/ml chitosan solution in a solution containing cancer cells, and all cancer cells die after 24 hours. It inhibits bacterial activity: Chitosan is easily dissolved in weak acid solvents. It is particularly worth noting that the dissolved solution contains amino groups (NH2+), which bind negative electrons to inhibit bacteria. Chitosan inhibits bacterial activity and makes it widely used in medicine, textiles and food. Chitosan and its derivatives have good antibacterial activity and can inhibit the growth and reproduction of some fungi, bacteria, and viruses. As of 2013, there are three possible mechanisms: one is due to the polycation of chitosan, which is easy to act on the surface of the fungal cell with negatively charged groups, thus changing the fluidity and permeability of the pathogen cell membrane; Replication and transcription; the third is to block the metabolism of pathogens. Since 2010, many researchers have proposed that chitosan can achieve antibacterial goals by inducing pathogenesis-related proteins, accumulating secondary metabolites and signaling. The effect of water-soluble chitosan such as chitosan lactate, chitosan glutamate and chitosan hydrogenated glutamate on different bacterial cultures. It was found that chitosan lactate and chitosan glutamate have high antibacterial activity against Gram-positive bacteria and Gram-negative bacteria.
具有免疫调节活性Immunomodulatory activity
壳聚糖具有激活机体***、介导机体***的系列生物学效应，提高吞噬细胞的***功能。巨噬细胞表面存在着细菌多糖的受体，而壳聚糖作为细菌多糖的类似物，能刺激巨噬细胞活化，产生如下反应：促进其吞噬功能，增强它在其它免疫应答中的协同效应，从而实现机体对T细胞、NK细胞和B细胞的调节，介导机体的细胞免疫应答和体液免疫应答。因此，壳聚糖具有对机体的免疫调节作用。Chitosan has a series of biological effects that activate the body system and mediate the body system, and improve the system function of phagocytic cells. The receptor for bacterial polysaccharides exists on the surface of macrophages, and chitosan, as an analogue of bacterial polysaccharides, stimulates the activation of macrophages, producing the following reactions: promoting its phagocytic function and enhancing its synergistic effect in other immune responses, Thereby, the body regulates T cells, NK cells and B cells, and mediates the cellular immune response and humoral immune response of the body. Therefore, chitosan has an immunomodulatory effect on the body.
癌症患者服用壳聚糖后，可激活体内具有免疫功能的淋巴细胞，使其能分辨正常细胞和癌细胞，并杀死癌细胞。壳聚糖能调节体内的pH值到弱碱性，提高胰岛素的利用率，有利于防治糖尿病。此外，它还具有调节内分泌***的功能，使胰岛素分泌正常，抑制血糖升高，降低血脂。After taking chitosan, cancer patients can activate immune lymphocytes in the body to distinguish normal cells and cancer cells and kill cancer cells. Chitosan can regulate the pH value in the body to weakly alkaline, improve the utilization of insulin, and help prevent and treat diabetes. In addition, it also has the function of regulating the endocrine system, which makes insulin secretion normal, inhibits blood sugar and lowers blood lipids.
Figure PCTCN2016000544-appb-000002
Figure PCTCN2016000544-appb-000002
壳聚糖或其衍生物作为新佐剂成分的可行性：Feasibility of chitosan or its derivatives as a new adjuvant ingredient:
在皮氨钙新佐剂中以壳聚糖或其衍生物取代卡那霉素可以：1.从上述结构式可以看到壳聚糖或其衍生物带多个正电荷氨基，与PIC是大分子的聚阴离子结合，形成较卡那霉素更稳定PIC双链RNA结构，在灵长类和人体中更有效。2.安全性高，又去到了抗生素副作用。3.可口服，可注射。4.与佐剂性质相似，具有增强免疫，抗肿瘤功效。In the new preparation of sulphate calcium, the substitution of chitosan or its derivative for kanamycin can be: 1. From the above structural formula, it can be seen that chitosan or its derivative has multiple positively charged amino groups, and PIC is a macromolecule. The combination of polyanions forms a more stable PIC double-stranded RNA structure than kanamycin, and is more effective in primates and humans. 2. High safety, and went to the side effects of antibiotics. 3. Can be taken orally, can be injected. 4. Similar to the adjuvant, it has enhanced immunity and anti-tumor effect.
二、皮氨钙佐剂配方Second, the skin calcium calcium adjuvant formula
结合上述举例非抗生素类氨基化合物性质、安全性，写出皮氨钙佐剂配方，检定内容，意在说明在皮氨钙佐剂中以非胺基抗生素类氨基化合物取代卡那霉素的具体技术方案，说明实施的可行性，但并不限定不可更改。In combination with the above-mentioned examples of the nature and safety of non-antibiotic amino compounds, the formula of the sulphate adjuvant is written, and the content of the test is intended to illustrate the specific substitution of the non-amino antibiotic amino compound for the kanamycin in the sulphate adjuvant. The technical solution explains the feasibility of the implementation, but it is not limited and cannot be changed.
原料：raw material:
聚胞苷酸(Poly C)：可购买。Polycyanoic acid (Poly C): available for purchase.
聚肌苷酸(Poly I)：可购买。Poly inosine (Poly I): available for purchase.
非抗生素类氨基化合物：可购买Non-antibiotic amino compounds: available for purchase
氯化钙：可购买Calcium chloride: can be purchased
制备：按聚肌胞(PIC)1.25mg/ml制备Preparation: Prepared according to poly myocyte (PIC) 1.25 mg/ml
1、0.006M PBS 50 000ml pH8.41, 0.006M PBS 50 000ml pH8.4
0.85％Nacl：425g溶解在50 000ml注射用水中。0.85% Nacl: 425 g was dissolved in 50 000 ml of water for injection.
Na2HPO4l2H2O：107.44g溶解在49 000ml 0.85％Nacl中。Na 2 HPO 4 l2H 2 O: 107.44 g was dissolved in 49 000 ml of 0.85% NaCl.
NaH2PO4.2H2O 0.936g：107.44g溶解在1 000ml 0.85％Nacl中。NaH 2 PO 4 .2H 2 O 0.936 g: 107.44 g was dissolved in 1 000 ml of 0.85% NaCl.
2、皮氨钙佐剂(20080701)配制：40 000ml，按40400ml投料(另加200ml Cacl2，并调pH)。2, sulphate adjuvant (20080701) preparation: 40 000ml, according to 40400ml feed (plus 200ml Cacl2, and adjust the pH).
原料比例批号理论用量含量实际用量PBS(ml)Raw material ratio batch number theoretical dosage content actual dosage PBS (ml)
Figure PCTCN2016000544-appb-000003
Figure PCTCN2016000544-appb-000003
非抗生素氨基化合物待定1000Non-antibiotic amino compounds to be determined 1000
补充PBS至40 000ml，另加：0.1M Cacl2(衢州巨化试剂有限公司20060401)：2.222g 200ml注射用水，用Na2HPO4.12H2O约100ml调pH至7.2。所有溶质分别溶解，45摄氏度30分，滴加0.1M Cacl2，调pH，0.22um滤膜过滤，分装。PBS was added to 40 000 ml, plus: 0.1 M Cacl 2 (Cangzhou Juhua Reagent Co., Ltd. 20060401): 2.222 g of 200 ml water for injection, and adjusted to pH 7.2 with about 100 ml of Na 2 HPO 4.12H 2 O. All solutes were dissolved, 45 ° C for 30 minutes, 0.1 M CaCl 2 was added dropwise, pH was adjusted, filtered through a 0.22 um filter, and dispensed.
三、检定Third, the verification
举例写出皮氨钙佐剂需检定的内容，意在表明实施的可行性，但并不限定不可更改。For example, the content of the picocalcium adjuvant to be tested is intended to indicate the feasibility of the implementation, but it is not limited and cannot be changed.
1、皮氨钙佐剂成品检定：检验依据：国家聚肌胞注射液检定标准WS1-XG-050-20001. Determination of the product of sulphate-adjuvant adjuvant: test basis: National concentrating cell injection test standard WS1-XG-050-2000
皮氨钙佐剂成品检定项目 Skin calcium calcium adjuvant finished product verification project
Figure PCTCN2016000544-appb-000004
Figure PCTCN2016000544-appb-000004
结论：皮氨钙佐剂各项检定结果必须符合WS1-XG-050-2000及含有该佐剂疫苗相关检定的规定。Conclusion: The results of the determination of the sulphate adjuvant must comply with the WS1-XG-050-2000 and the relevant regulations for the vaccine containing the adjuvant.
2.小白鼠重复毒性检定：为进一步检定毒性，需要在小白鼠重复注射5-6针，观察。不能死亡，并应体重增加。小白鼠***反应实验。2. Repeated toxicity test in mice: For further testing of toxicity, it is necessary to repeat 5-6 needles in mice for observation. Can't die, and should gain weight. Mouse allergy experiment.
3.猴体试验：包括安全性、有效性。3. Monkey test: including safety and effectiveness.
五、皮氨钙佐剂疫苗的制备V. Preparation of a picocalcium adjuvant vaccine
根据上述皮氨钙佐剂的制备，举例写出皮氨钙佐剂狂犬病疫苗制备、检定方案，意在使人们更加理解制备皮氨钙佐剂疫苗实施的可行性，绝不是仅限于皮氨钙佐剂狂犬病疫苗。皮氨钙佐剂疫苗包括所有含有该佐剂的疫苗，特别是细胞内感染疫苗和肿瘤疫苗。According to the preparation of the above-mentioned slaked calcium calcium adjuvant, the preparation and verification scheme of the rabies vaccine of squamous calcium adjuvant is exemplified, and the intention is to make people understand the feasibility of preparing the vaccine for the preparation of the squamous calcium adjuvant vaccine, which is not limited to the skin of calcium sulphate. Adjuvant rabies vaccine. The picocalcium adjuvant vaccine includes all vaccines containing the adjuvant, particularly intracellular infection vaccines and tumor vaccines.
皮胺钙佐剂狂犬病纯化疫苗(地鼠肾细胞)Peptide calcium adjuvant rabies purified vaccine (hamster kidney cells)
原料：raw material:
1.皮氨钙佐剂：总量：7200ml。1. Skin calcium calcium adjuvant: total: 7200ml.
2.狂犬病纯化疫苗原液(地鼠肾细胞)：2. Rabies purified vaccine stock solution (hamster kidney cells):
由生产厂家提供浓缩纯化疫苗原液1800ml(已加20％人白蛋白37.5ml，0.5％硫柳汞1.8ml。1800ml of concentrated purified vaccine stock solution is provided by the manufacturer (added 20% human albumin 37.5ml, 0.5% thimerosal 1.8ml).
制造方法Production method
皮氨钙佐剂狂犬病纯化疫苗的制备Preparation of purified vaccine for rabies adjuvant rabies
1.取1800ml浓缩的人用狂犬病纯化疫苗原液与皮胺钙佐剂7200ml复合。1. Take 1800 ml of concentrated human rabies purified vaccine stock solution and 7200 ml of dermatan calcium adjuvant.
2.按0.3-0.4％加20％人白蛋白100ml，加5％硫柳汞7.2ml，加谷胺酸钠 (10％)90ml。2. According to 0.3-0.4% plus 20% human albumin 100ml, add 5% thimerosal 7.2ml, add sodium glutamate (10%) 90ml.
3.分装：按1ml/安瓶/剂量分装，可制备出皮氨钙狂犬病纯化疫苗1600人份。3. Packing: According to 1ml / ampere / dose, a sample of 1600 human rabies rabies purified vaccine can be prepared.
3.狂犬病纯化疫苗纯化后原液的制备(供检定用)3. Preparation of stock solution after purification of rabies purified vaccine (for verification)
取2ml不含白蛋白的狂犬病纯化疫苗浓缩液与8ml PBS混合，按该比例共配制30ml。2 ml of albumin-free rabies purified vaccine concentrate was mixed with 8 ml of PBS, and 30 ml of the mixture was prepared in this ratio.
4.狂犬病纯化疫苗纯化后原液刺激抗原的制备(供细胞免疫检定用)4. Preparation of stimulating antigen after purification of rabies purified vaccine (for cellular immunoassay)
取2ml不含人白蛋白和硫柳汞的狂犬病纯化疫苗浓缩液30ml，蛋白含量约120ug，0.5ml/Apendouf50管，低温保存。Take 2ml of rabies purified vaccine concentrate containing no human albumin and thimerosal, 30ml, protein content of about 120ug, 0.5ml/Apendouf50 tube, and store at low temperature.
检定结果Verification result
检验依据：国家药典狂犬病纯化疫苗制造检定审定规程以及皮氨钙佐剂疫苗特殊检定要求(待定)Test basis: National Pharmacopoeia rabies purified vaccine manufacturing verification procedures and special requirements for serotonin adjuvant vaccine (to be determined)
皮氨钙佐剂狂犬病纯化疫苗检定报告单Dermium Calcium Adjuvant Rabies Purification Vaccine Verification Report
Figure PCTCN2016000544-appb-000005
Figure PCTCN2016000544-appb-000005
各项检定结果应完全符合各项规定，合格。The results of each verification shall be in full compliance with the provisions and qualified.
以上所述仅为本发明的实施例，并非因此限制本发明的专利范围，凡是利用本发明举例说明非抗生素胺基类化合物及皮胺钙佐剂配方、检定内容和皮胺钙佐剂疫苗、检定内容所作的数据或等效流程变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围内。 The above description is only an embodiment of the present invention, and thus does not limit the scope of the patent of the present invention, and the present invention is exemplified by the non-antibiotic amine-based compound and the dermatan calcium adjuvant formulation, the test content, and the dermatan calcium adjuvant vaccine. The data or equivalent process transformation of the verification content, or directly or indirectly applied to other related technical fields, is equally included in the scope of patent protection of the present invention.

Claims (5)

  1. 一种含氨基化合物(除外氨基类抗生素)和钙离子的聚肌苷酸聚胞苷酸(聚肌胞)在制备免疫佐剂中的应用。该佐剂简称：皮氨钙佐剂。成分中文简写为：聚肌胞-氨基化合物-Cacl2。The use of a polyinosinic acid polycyanoic acid (polymeric myocyte) containing an amino compound (excluding an amino antibiotic) and calcium ions for the preparation of an immunoadjuvant. The adjuvant is referred to as: a picocalcium adjuvant. The Chinese compound is abbreviated as: poly-cell-amino compound-Cacl 2 .
  2. 根据权利1所述的一种含有皮氨钙佐剂的疫苗。包括细胞内感染(病毒和细胞内感染的细菌、寄生虫等)疫苗和肿瘤疫苗用于预防和治疗。其特征在于所述的佐剂含有氨基化合物(除外氨基类抗生素)和钙离子的聚肌苷酸聚胞苷酸(聚肌胞)。A vaccine containing a calcium sulphate adjuvant according to claim 1. Vaccines and tumor vaccines including intracellular infections (bacteria, parasites, etc. of viruses and intracellular infections) are used for prevention and treatment. It is characterized in that the adjuvant contains an amino compound (excluding an amino antibiotic) and a calcium ion polyinosinic polycytidine (poly-cell).
  3. 一种含氨基化合物(除外氨基类抗生素)和钙离子的聚肌苷酸聚胞苷酸(聚肌胞)的免疫佐剂在制备用于哺乳动物或人的预防性和治疗性疫苗中的应用。Use of an immunoadjuvant of polyinosinic poly(cytidine) containing amino compounds (excluding amino antibiotics) and calcium ions for the preparation of prophylactic and therapeutic vaccines for mammals or humans .
  4. 根据权利1所述用含有皮氨钙佐剂的免疫刺激物用于体外刺激免疫细胞，回输体内，以达到增强免疫抗肿瘤、抗细胞内感染效果的应用。其特征在于所述的免疫刺激物含有氨基化合物(除外氨基类抗生素)和钙离子的聚肌苷酸聚胞苷酸(聚肌胞)。The immunostimulant containing the sulphate adjuvant is used according to claim 1 for stimulating immune cells in vitro and returning to the body to achieve an effect of enhancing the anti-tumor and anti-intracellular infection effects of the immune system. It is characterized in that the immunostimulant contains an amino compound (excluding an amino antibiotic) and a calcium ion polyinosinic polycytidine (polymeric myocyte).
  5. 根据权利1所述用皮氨钙佐剂为免疫增强剂，非特异促进机体免疫力的应用。其特征在于所述的免疫增强剂含有氨基化合物(除外氨基类抗生素)和钙离子的聚肌苷酸聚胞苷酸(聚肌胞)。 The use of the sulphate adjuvant as an immunopotentiator according to claim 1 for non-specific promotion of immunity of the body. It is characterized in that the immunopotentiator contains an amino compound (excluding an amino antibiotic) and a calcium ion polyinosinic polycytidine (polymeric myocyte).
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